The 19-base oligonucleotide probe was chemically synthesized and modified from Shanghai Shangon Bioengineering Co., Ltd. The ASON sequence was designed as 5′- AATTCTTAAATTGGGCTGG -3′, which was completely complementary to the HOTAIR fragment, and the ASONM（mismatched antisense oligonucleotides） sequence was 5′-AATACTTAGATTAGGCAGG-3′ (The underlined part is the substituted nucleosides).Two probes were modified with 2' methylation at both ends of the sequence and two bases at each end were thiomodificed to improve the stability. A primary amine structure is connected at the 3 'end, and (CH2)6 is used as the connector between the NH2 and the skeleton. Ultimately, the synthetic structure was 5’-CH2 ASON/ASONM-(CH2)6-NH2-3'. 99mTc is obtained from the 99mTc radionuclide generator produced by China Atomic Energy Research Institute.
Synthesis and labeling of the probe
4OD ASON dissolved in 50μl buffer (2mol/l NaCl, 0.5mol/l NaHCO3, 2mmol/l EDTA), HYNIC (TriLink, US) dissolved in DMF solution (10mg/ml). Then mixed at a molar ratio of 25:1( HYNIC: ASON) and avoid light for 1 h. Then added 60% methanol to the total volume of 500μl, using ultrafilter tube(Sartorius, GER) 15000g/rcf to centrifuge for 10min (ensure that the volume after centrifugation was less than 50μl). Next, added 100μl Tricine(100mg/ml), 30μl 99mTc(222MBq) as well as 4μl fresh SnCl2•2H2O (1mg/ml) to the above reactants in turn, reacted for 60min. After the reaction, using Sephadex G25(GE, US) to separate and purify. 15 tubes of eluent were collected, then measured the radioactivity counts and nucleic acid concentration of each tube. Take the peak tube for the following up experiment.
Fresh human serum is provided by volunteers in our department. The institutional review committee of the General Hospital of Ningxia Medical University approved the study and all the volunteers obtained informed consent. And this experiment is carried out in accordance with the Helsinki Declaration. 99mTc-HYNIC-ASON was incubated in saline and fresh human serum at 37℃ and room temperature, respectively (the volume ratio of probe to serum / saline was 1:1). The radiochemical purity was detected by thin layer paper chromatography (iTLC) at 0, 2, 4, 6, 8, 12 h.
Agarose gel electrophoresis
To identify the integrity of probes and eliminate the degradation of antisense oligonucleotides after labeling. 1% agarose gel was configured, followed by unbonded ASON sample, 99mTc, 99mTc-HYNIC-ASON before and after purification. The voltage was 120V, electrophoresis for 20 minutes, then the band was observed under UV.
Cell culture and transfection.
U87 glioma cells were purchased from Chinese Academy of Sciences, and cultured in DMEM(Invitrogen,US) medium containing 15% fetal bovine serum and 1% antibiotics, in a CO2 incubator at 37 ℃ for 24 hours, then passaged when the cell density reached 90%.
For transfection (Lip-99mTc-HYNIC-ASON and Lip-99mTc-HYNIC-ASONM), 10μg purified 99mTc-HYNIC-ASON/ 99mTc-HYNIC-ASONM were added to 500μl DMEM without serum and antibiotics; 25μl Lipofectamine 2000 (Invitrogen,US) were added to 475μl DMEM, and the mixtures were placed at room temperature for 5min respectively, then mixed two of them for 20min. After added liposome-coated 99mTc-HYNIC-ASON/ 99mTc-HYNIC-ASONM to the walls and cultured in a 37℃ incubator for 6 hours, the medium was replaced by DMEM contained 15% fetal bovine serum for 24-48 hours to complete transfection.
U87 cells were inoculated in 12-well plate at 1 × 10 5 density and cultured overnight in DMEM containing 15%FBS without antibiotics. The cells were divided into liposome transfected and non-transfected group. In the transfection group, 200μl DMEM, 500ng 99mTc-HYNIC-ASON or 99mTc-HYNIC-ASONM (37kBq) and 3μl Lipofectamine 2000 were added into each well, 500ng 99mTc-HYNIC-ASON or 99mTc-HYNIC-ASONM (37kBq) in the non-transfection group. After cultured in 37 ℃ incubator, each well of culture medium was collected and washed three times with 100μl PBS collected into the same EP tube, labeld Cout at 0.5h,1h, 2h, 4h, and 6h. Then the cell was collected with trypsin containing EDTA, 100μl PBS in each well was washed three times to EP tube labeled Cin. The radioactivity counts of Cin and Cout were measured by γ radioimmunoassay counter(Chinese Academy of Metrology), and the uptake rate of cells to the probe at each time was calculated. Calculation formula: cell uptake rate = Cin/ (Cin+Cout) × 100%.
Animal Xenograft Model
BALB/c nu/nu mice (female, weight ±SD, 206.0g, age 3~4wk) were fed in the Experimental Animal Center of Ningxia Medical University. Malignant glioma U87 cells (5×1011) were subcutaneously injected into the right fore axilla of each mouse. When the diameter of the tumor reached 1.0-1.5cm, it was used in the follow-up experiment. All animal experiments have passed the ethical review of the Animal Experimental Center of Ningxia Medical University and are conducted in accordance with the guidelines of the Animal Welfare Committee.
Twenty nude mice were randomly divided into 5 groups with 4 mice in each group. Lip-99mTc-HYNIC-ASON 1μg, 2.59MBq (100μl) was injected into the tail vein. Then the mice were killed by cervical dislocation at 1, 2, 4, 6 and 8 hours after 100μl of blood was taken from ophthalmic vein. After that, tissues like Heart, liver, spleen, kidney, stomach, small intestine, bladder, muscle, bone and tumor were removerd and weighed, then radioactivity count was measured. The distribution results were recorded as the percentage of radioactivity per gram of tissue(% ID/g).
Images were performed by a SPECT scanner at 1h, 2h, 4h, 6h and 8h respectively，after 4μg, 14.8MBq (150μl) Lip-99mTc-HYNIC-ASON/ 99mTc-HYNIC-ASONM probe were injected into the tail vein. For blocking groups, 10μg liposome transfected unlabeled probes were injected 2 hours before Lip-99mTc-HYNIC-ASON injection.The collection counts was 100 KCous and stored as a 64×64 matrix at 3.2 zoom, then T/M (Tumor/Muscle) and T/A (Tumor/Abdomen) ratio of regions of interest were calculated.
Establish a database, all data are processed by SPSS22.0 statistical software, variables are represented by average±SD. The statistical comparison between variables was carried out by analysis of variance (ANOVA). P<0.05 is considered to be statistically significant.